ENZITEC 2012 – X th Brazilian Seminar on Enzyme Technology – 7 th –10 th of October, 2012, Blumenau, SC, Brazil. BIOPROSPECTION OF LOW MASS PROTEASE INHIBITORS FROM AMAZON BIODIVERSITY USING SURFACE PLASMON RESONANCE TECNOLOGY Nelice M. B. Serbino 1 , José Roniele do N. Monteiro 1,3 , Leandro S. M. Dill 1 , Gustavo H. M. F. Souza 2 , Rodrigo G. Stábeli 1,3 , Leonardo A. Calderon 1,3* 1 Centro de Estudos de Biomoléculas Aplicadas a Saúde - CEBio, Fiocruz Rondônia, 76812-245 Porto Velho, RO, Brazil; 2 MS Applications Development Laboratory, Waters Technologies Brazil, 06455-020 Alphaville, SP, Brazil, 3 Departamento de Medicina, Universidade Federal de Rondônia, 76800-000 Porto Velho, RO, Brazil.*e-mail: calderon@fiocruz.br Keywords: Inga, chymotrypsin, peptide inhibitor INTRODUCTION Amazonian plant species are a treasure store of bioactive molecules, such as those belonging to the genus Inga, with next to three hundred species, which are a huge source of protease inhibitors. Surface Plasmon Resonance (SPR) is a technique used in high-throughput screening of binders to a validated target using a biosensor (1) being widely used by pharmaceutical industry (2). In order to discover low mass protease inhibitors from Amazonian biodiversity with high potential for the development of anticancer drugs, antiviral and protozoa drugs (3), this study resulted in the isolation and characterization of a new low mass chymotrypsin inhibitor from Inga edulis seeds, named as Inga chymotrypsin inhibitor peptide - ICIP. RESULTS AND DISCUSSION Seeds collected at Cruzeiro do Sul-AC were powdered and extracted with water/alcohol solution during 16hs. Extracts were dried and assayed against trypsin and chymotrypsin using N- benzoyl-L-arginine-p-nitroanilide and N-glutaryl-L- phenylalanyl-p-nitroamilide, as chromogenic substrates respectively. The extract obtained with ethanol 90% showed high chymotrypsin inhibitory activity (Fig. 1A). SPR binding assay with recovery, performed with BIACORE T200 (GE Healthcare), confirmed the presence of a high affinity interaction with chymotrypsin (Fig. 1B). Figure 1. A) Ethanol extracts inhibitory activity against trypsin (blue) and chymotrypsin (red); B) ETOH 90% extract SPR sensorgram. Mass spectrometry analysis of recovered sample was performed using a Xevo G2 QTof (Waters, Manchester, UK). MS/MS acquisition in positive mode of the precursor ion with 1.106,46 m/z was obtained by collision-induced dissociation (CID) at the T-Wave collision cell filled with argon gas (Fig. 2A). De novo sequence was performed using MaxEnt 3 software (Waters, Manchester, UK) for MS/MS de-convolution and PepSeq application for interpretation of y + and b + ions. The sequence obtained was HAY(*)NVT with 1.106,46 (M+H) + (Fig. 2B). The peptide was named as Inga Chymotrypsin Inhibitor Peptide – ICIP. Figure 2. A) MS/MS acquisition of the 1.106,46 m/z precursor ion*; B) Structure of ICIP* *Information is partially protected due to ICIP potential biotechnological application and publication pending. CONCLUSION ICIP is a new natural peptide, potentially the first member of new inhibitory peptide family. Due to its low mass, net charge and high affinity to chymotrypsin, it has potential anticancer activity, however, further biological assays are in progress. ACKNOWLEDGEMENTS FINEP, CNPq, CAPES, BIONORTE, Inpetam REFERENCES 1 Rich, R. L., and Myszka, D. G. (2006) Survey of the year 2005 commercial optical biosensor literature. J. Mol. Recognit. 19, 478–534. 2 Karlsson, R., Kullman-Magnusson, M., Hämäläinen, M.D., Remaeus, A., Andersson, K., Borg, P., Gyzander, E., and Deinum, J. 2000. Biosensor analysis of drug–target interactions: Direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors. Anal. Biochem. 278: 1–13. 3 Craik, M. S.; Debouck, C. Proteases as Therapeutic Targets. In Perspectives in Drug Discovery and Design; McKerrow, J. H., James, M. N. G., Eds.; ESCOM: Leiden, 1995; Vol. 2, pp 1-12. View publication stats View publication stats